Fluorimetric determination of monobromobimane and o-phthalaldehyde adducts of γ-glutamylcysteine and glutathione: application to assay of γ-glutamylcysteinyl synthetase activity and glutathione concentration in liver

The reversed-phase HPLC separation of fluorescent o-phthalaldehyde (OPA) derivatives has been applied to the assay of hepatic γ-glutamylcysteine and glutathione (GSH) levels and the enzymes producing these peptides. The method has been compared to the assay using monobromobimane (MB) as the derivatizing agent. The OPA method has the advantage of faster derivitization, the lack of need to adjust the pH, isocratic separation and selectivity for GSH and γ-glutamylcysteine. The MB method requires pH adjustment following derivatization and gradient elution chromatography. MB is also non-selective, yielding fluorescent derivatives of all biological thiols and more interfering peaks on the chromatogram. MB-based analyses are also approximately sixty times more expensive per sample. MB yields fluorescent degradation products on exposure to light. OPA adducts are stable for up to ten days when stored at −20°C. OPA detection is sensitive to 12.5 pmol in the sample, at a signal-to-noise ratio of 2.5. The two methods correlate well. Hepatic γ-glutamylcysteine synthetase in the same liver preparation was found to be 4.85±0.47 nmol min⁻¹ mg⁻¹ protein by the OPA method and 4.42±0.52 nmol min⁻¹ mg⁻¹ protein by the MB method. GSH concentrations were found to be 90.4±6.5 nmol/mg protein for the OPA method and 92.5±3.4 for the MB method.     

Determination of biological thiols by high-performance liquid chromatography following derivatization by ThioGlo maleimide reagents

The importance of thiols has stimulated the development of a number of methods for determining glutathione and other biologically significant thiols. Methods that are currently available, however have some limitations, such as being time consuming and complex. In the present study, a new high-performance liquid chromatography (HPLC) method for determining biological thiols was developed by using 9-Acetoxy-2-(4-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)phenyl)-3-oxo-3H-naphtho[2,1-b]pyran (ThioGlo™3) as a derivatizing agent. ThioGlo™ reacts selectively and rapidly with the thiols to yield fluorescent adducts which can be detected fluorimetrically (λ_ex=365 nm, λ_em=445 nm). The within-run coefficient of variation for glutathione (GSH) by this method ranges from 1.08 to 2.94% whereas the between-run coefficient of variation for GSH is 4.31–8.61%. For GSH, the detection limit is around 50 fmol and the GSH derivatives remain stable for 1 month, if kept at 4°C. Results for GSSG and cysteine are also included. The ThioGlo™ method is compared to our previous method in which N-(1-pyrenyl)maleimide (NPM) is used to derivatize thiol-containing compounds. The present method offers various advantages over the currently accepted techniques, including speed and sensitivity.     

para-sulfobenzoyloxybromobimane: a new membrane-impermeable reagent useful for the analysis of thiols and their export from cells.

para-Sulfonylbenzoyloxybromobimane (sBBr) was shown to be similar to the fluorescent labeling agent monobromobimane (mBBr) in reacting rapidly and selectively with thiols to produce stable derivatives which are readily separated by HPLC. Chromatography of the sBBr derivative provides a useful means of confirming the identification of an unknown thiol based upon the chromatography of its mBBr derivative and can be useful for quantitative determination of polycationic thiols for which chromatography of the mBBr derivative is unsatisfactory. Unlike mBBr, which readily penetrates cells, sBBr was found not to be taken up by cells. These characteristics allow sBBr to be used, in conjunction with mBBr, to quantify the export of thiols from cells, as illustrated for GSH and the radioprotective drug WR1065, from V79 cells. Simultaneous determination of GSH and glutathione disulfides in cell culture medium could be achieved by labeling of thiols with sBBr followed by reduction of disulfides with dithiothreitol, labeling of the resulting thiols with mBBr, and HPLC analysis for both glutathione derivatives.     

High-performance liquid chromatographic assay of hydroxyl free radical using salicylic acid hydroxylation during in vitro experiments involving thiols

A HPLC method was developed to monitor the production of hydroxyl free radical (°OH) produced during in vitro experiments: (i) a chemical reaction involving EDTA chelated ferric ion and various exogenous and endogenous thiols [glutathione (GSH) and its metabolites], and (ii) an enzymatic reaction corresponding to the breakdown of GSH catalyzed by γ-glutamyltransferase (GGT). The method relies upon the use of a selective trapping reagent of °OH: salicylic acid (SA). The three resulting dihydroxylated products, i.e., 2,3-dihydroxybenzoic acid (DHB), 2,5-DHB and catechol, were measured in an ion-pairing reversed-phase HPLC system coupled with amperometric detection; the sum of the three concentrations was used to quantify the production of °OH during in vitro experiments. Resulting data demonstrate that °OH is produced during Fenton-like reactions involving thiols and GSH catabolism via GGT.     

Effect of Cadmium on gamma-Glutamylcysteine Synthesis in Maize Seedlings

Cysteine, γ-glutamylcysteine, and glutathione and the extractable activity of the enzymes of glutathione biosynthesis, γ-glutamylcysteine synthetase (EC 6.3.2.2) and glutathione synthetase (EC 6.3.2.3), were measured in roots and leaves of maize seedlings (Zea mays L. cv LG 9) exposed to CdCl₂ concentrations up to 200 micromolar. At 50 micromolar Cd²⁺, γ-glutamylcysteine contents increased continuously during 4 days up to 21-fold and eightfold of the control in roots and leaves, respectively. Even at 0.5 micromolar Cd²⁺, the concentration of γ-glutamylcysteine in the roots was significantly higher than in the control. At 5 micromolar and higher Cd²⁺ concentrations, a significant increase in γ-glutamylcysteine synthetase activity was measured in the roots, whereas in the leaves this enzyme activity was enhanced only at 200 micromolar Cd²⁺. Labeling of isolated roots with [³⁵S]sulfate showed that both sulfate assimilation and glutathione synthesis were increased by Cd. The accumulation of γ-glutamylcysteine in the roots did not affect the root exudation rate of this compound. Our results indicate that maize roots are at least in part autonomous in providing the additional thiols required for phytochelatin synthesis induced by Cd.     

Manipulation of Glutathione and Amino Acid Biosynthesis in the Chloroplast

Poplars (Populus tremula × Populus alba) were transformed to overexpress Escherichia coli γ-glutamylcysteine synthetase (γ-ECS) or glutathione synthetase in the chloroplast. Five independent lines of each transformant strongly expressed the introduced gene and possessed markedly enhanced activity of the gene product. Glutathione (GSH) contents were unaffected by high chloroplastic glutathione synthetase activity. Enhanced chloroplastic γ-ECS activity markedly increased γ-glutamylcysteine and GSH levels. These effects are similar to those previously observed in poplars overexpressing these enzymes in the cytosol. Similar to cytosolic γ-ECS overexpression, chloroplastic overexpression did not deplete foliar cysteine or methionine pools and did not lead to morphological changes. Light was required for maximal accumulation of GSH in poplars overexpressing γ-ECS in the chloroplast. High chloroplastic, but not cytosolic, γ-ECS activities were accompanied by increases in amino acids synthesized in the chloroplast. We conclude that (a) GSH synthesis can occur in the chloroplast and the cytosol and may be up-regulated in both compartments by increased γ-ECS activity, (b) interactions between GSH synthesis and the pathways supplying the necessary substrates are similar in both compartments, and (c) chloroplastic up-regulation of GSH synthesis is associated with an activating effect on the synthesis of specific amino acids formed in the chloroplast.     

Quantitation of reduced and total glutathione at the femtomole level by high-performance liquid chromatography with fluorescence detection: application to red blood cells and cultured fibroblasts

A new rapid and highly sensitive HPLC method with ortho-phthalaldehyde (OPA) pre-column derivatization has been developed for determination of reduced glutathione (GSH) and total glutathione (GSHt) in human red blood cells and cultured fibroblasts. OPA derivatives are separated on a reversed-phase HPLC column with an acetonitrile–sodium acetate gradient system and detected fluorimetrically. An internal standard (glutathione ethyl ester) is added to facilitate quantitation. Total glutathione is determined after reduction of disulfide groups with dithiothreitol; the oxidized glutathione (GSSG) concentration is calculated by subtraction of the GSH level from the GSHt level. The assay shows high sensitivity (50 fmol per injection, the lowest reported), good precision (C.V. <5.0%), an analytical recovery of GSH and GSSG close to 100%, and linearity (r>0.999). This HPLC technique is very simple and rapid. Its wide applicability and high sensitivity make it a convenient and reliable method for glutathione determination in various biological samples.     

Determination of S-nitrosoglutathione in human and rat plasma by high-performance liquid chromatography with fluorescence and ultraviolet absorbance detection after precolumn derivatization with o-phthalaldehyde.

An analytical method is described for the quantification of S-nitrosoglutathione (GSNO), a potent physiological vasodilator and inhibitor of platelet aggregation, in the presence of a high excess of reduced glutathione (GSH). The method is based on the quantitative elimination of GSH by N-ethylmaleimide, the conversion of GSNO by 2-mercaptoethanol to GSH, its reaction with o-phthalaldehyde (OPA) to form a highly fluorescent and UV-absorbing tricyclic isoindole derivative, and subsequent high-performance liquid chromatographic (HPLC) separation with fluorescence and/or UV absorbance detection. The OPA derivatives of GSH and GSNO obtained by this method were found to be identical by mass spectrometry. GSH (up to 50 microM) did not interfere with the analysis of GSNO (up to 1000 nM). The limits of detection of the method for buffered aqueous solutions of GSNO were determined as 3 nM using fluorescence and 70 nM using UV absorbance detection. Isolation of GSNO by HPLC analysis (pH 7.0) of plasma ultrafiltrate samples (200 microl) prior to derivatization allows specific and artifact-free quantification of GSNO in human and rat plasma. Reduced and oxidized glutathione, nitrite, and cysteine did not interfere with the measurement of GSNO in human and rat plasma. The limit of quantitation (LOQ) of the combined method was determined as 100 nM of GSNO in human plasma ultrafiltrate using fluorescence detection. No endogenous GSNO could be detected in ultrafiltrate samples of plasma of 10 healthy humans at concentrations exceeding the LOQ of the method. After iv infusion of GSNO (125 micromol/kg body wt) in a rat for 20 min GSNO and GSH were detected in rat plasma at 60 and 130 microM, respectively. The method should be useful to investigate formation, metabolism, and reactions of GSNO in vitro and in vivo at physiologically relevant concentrations.     

Analysis of biological thiols: derivatization with monobromotrimethylammoniobimane and characterization by electrophoresis and chromatography

A new method for analysis of biological thiols based upon their conversion to fluorescent derivatives by reaction with monobromotrimethylammoniobimane (qBBr) is described. The derivatives are separated by chromatography and by electrophoresis on cellulose thinlayer chromatography plates. The use of two-dimensional mapping makes it possible to differentiate between a wide variety of biological thiols including N-acetylcysteine, CoA, cysteine, cysteinylglycine, cysteamine, ergothioneine, glutathione, γ-glutamylcysteine, homocysteine, mercaptopyrimidine, pantetheine, 4′-phosphopanetheine, thiosulfate, and thiouracil. For applications to biological samples thiols were isolated from crude extracts by binding to a mercuriagarose gel. Following removal from the gel with dithiothreitol, the thiols were derivatized with qBBr. The methods were tested by showing that glutathione is the major thiol in human red blood cells, that glutathione and ergothioneine are the major thiols in Neurospora crassa conidia, and that Bacillus cereus vegetative cells lack glutathione but contain cysteine, pantetheine, and an unidentified thiol in significant amounts.     

Effects of a root-colonized dark septate endophyte on the glutathione metabolism in maize plants under cadmium stress

A high Cd-tolerant dark septate endophyte (DSE), Exophiala pisciphila, was inoculated into maize (Zea mays L.) roots under Cd stress. The Cd content, enzymes activity and thiol compound content relevant to glutathione (GSH) metabolism in maize leaves were analyzed. The Cd content in maize shoots increased with increasing Cd stress, but the DSE significantly reduced the Cd content at the 40?mg/kg Cd treatment. Cd stress increased the enzyme activity of glutathione reductase (GR), glutathione S-transferase (GST) and glutathione peroxidase (GSH-Px) as well as the thiol compound contents of sulfur, thiols (-SH) and oxidized glutathione (GSSG). The content of reduced GSH and the GSH/GSSG ratio reached a peak at the 5?mg/kg Cd treatment but then decreased with increasing Cd stress. Furthermore, the DSE significantly enhanced the GR and GSH-Px activity and increased the contents of -SH and GSH under low Cd stress (5 and 10?mg/kg), but decreased the γ-glutamylcysteine synthetase and GST activity under high Cd stress (20 and 40?mg/kg). Highly positive correlations between the Cd content with enzymes activity and enzymes activity with thiol compound content were observed. Results indicated that DSE played a role in activating GSH metabolism in maize leaves under Cd stress.     

Determining glutathione and glutathione disulfide using the fluorescence probe o-phthalaldehyde

Because of the importance of glutathione (GSH) and glutathione disulfide (GSSG) in cellular signal transduction, gene regulation, redox regulation, and biochemical homeostasis, accurate determination of cellular glutathione levels is critical. Several procedures have been developed, but many suffer from overestimating GSSG or from cellular substances interfering or competing with GSH determination. Assays based on HPLC, with enzymatic reduction of GSSG by glutathione reductase and NADPH, appear to be valid but are limited in sample throughput and availability of equipment. The fluorescence probe o-phthalaldehyde (OPA, phthalic dicarboxaldehyde) reacts with GSH and has a high quantum yield, yet its use has been limited due to unidentified interfering and fluorescence-quenching substances in liver. This paper describes assay conditions under which these limitations are avoided. By using a phosphate-buffered assay at lower pH, interference with nonspecific reactants is minimal. Since enzymatic reduction is not possible due to the reaction of OPA with NAD(P)H and other stronger reducing agents, leading to an overestimation of GSSG levels, dithionite was used to reduce GSSG. High sample throughput combined with sensitive (20-pmol limit of detection) and accurate determination of GSH and GSSG using OPA is achievable with any monochromatographic spectrofluorometer. Sample preparation and storage conditions are described that return the same levels of GSH and GSSG for at least 4 weeks.     

Glutathione in Synechocystis 6803: A closer look into the physiology of a ΔgshB mutant

Glutathione (GSH) is a low molecular weight thiol compound that plays many roles in photosynthetic organisms. We utilized a ΔgshB (glutathione synthetase) mutant strain as a tool to evaluate the role of GSH in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis 6803), a model photosynthetic organism. The ΔgshB mutant does not synthesize glutathione, but instead accumulates the GSH precursor, γ-glutamylcysteine (γ-EC), to millimolar levels. We found that γ-EC was sufficient to permit cellular proliferation during optimal conditions, but not when cells were exposed to conditions promoting oxidative stress. Furthermore, we found that many factors affecting growth rate and photosynthetic activities strongly influenced cellular thiol content. Here, we are providing some additional insights into the role of GSH and γ-EC in Synechocystis 6803 during conditions promoting oxidative stress.Key words: redox, reactive oxygen species, cyanobacteria, photosynthesis, photosystem I, photosystem II, methyl viologen, metal, cadmium, arsenate, selenate     

The evolution of glutathione metabolism in phototrophic microorganisms

Summary Of the many roles ascribed to glutathione (GSH) the one most clearly established is its role in the protection of higher eucaryotes against oxygen toxicity through destruction of thiol-reactive oxygen byproducts. If this is the primary function of GSH then GSH metabolism should have evolved during or after the evolution of oxygenic photosynthesis. That many bacteria do not produce GSH is consistent with this view. In the present study we have examined the low-molecular-weight thiol composition of a variety of phototrophic microorganisms to ascertain how evolution of GSH production is related to evolution of oxygenic photosynthesis. Cells were extracted in the presence of monobromobimane (mBBr) to convert thiols to fluorescent derivatives, which were analyzed by highpressure liquid chromatography. Significant levels of GSH were not found in the green bacteria (Chlorobium thiosulfatophilum andChloroflexus aurantiacus). Substantial levels of GSH were present in the purple bacteria (Chromatium vinosum, Rhodospirillum rubrum, Rhodobacter sphaeroides, andRhodocyclus gelatinosa), the cyanobacteria [Anacystis nidulans, Microcoleus chthonoplastes S.G., Nostoc muscorum, Oscillatoria amphigranulata, Oscillatoria limnetica, Oscillatoria sp. (Stinky Spring, Utah),Oscillatoria terebriformis, Plectonema boryanum, andSynechococcus lividus], and eucaryotic algae (Chlorella pyrenoidsa, Chlorella vulgaris, Euglena gracilis, Scenedesmus obliquus, andChlamydomonas reinhardtii). Other thiols measured included cysteine, -glutamylcysteine, thiosulfate, coenzyme A, and sulfide; several unidentified thiols were also detected. Many of the organisms examined also exhibited a marked ability to reduce mBBr to syn-(methyl,methyl)bimane, an ability that was quenched by treatment with 2-pyridyl disulfide or 5,5-bisdithio-(2-nitrobenzoic acid) prior to reaction with mBBR. These observations indicate the presence of a reducing system capable of electron transfer to mBBr and reduction of reactive disulfides. The distribution of GSH in phototrophic eubacteria indicates that GSH synthesis evolved at or around the time that oxygenic photosynthesis evolved.     

Effects of Lipopolysaccharides from Gram-Negative Bacteria on the Level of Thiols in Blood Platelets

Lipopolysaccharide (endotoxin, LPS) activates blood platelets and stimulates generation of free radicals in these cells. The mechanism of platelet activation induced by LPS is not known. The aim of the present study was to examine how glutathione (GSH) and other thiol-containing compounds are involved in the oxidative stress in blood platelets caused by LPS. The HPLC technique has been used on the analysis of non-protein thiols from human blood platelets treated with lipopolysaccharides of different Gram-negative bacteria (Proteus mirabilis, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa). Our results show that LPSs caused an increase (about 10%) of the level of reduced glutathione (GSH) and other nonprotein thiols such as cysteine (CSH) and cysteinylglycine (CGSH), whereas the total pool of these compounds was almost unchanged. LPS may react directly with thiols, since after incubation of LPSs with glutathione alone (in reduced form) we observed a distinct decrease of the level of platelet GSH.     

Cadmium-Responsive Thiols in the Ectomycorrhizal Fungus Paxillus involutus

Molecular and cellular mechanisms underlying the sustained metal tolerance of ectomycorrhizal fungi are largely unknown. Some of the main mechanisms involved in metal detoxification appear to involve the chelation of metal ions in the cytosol with thiol-containing compounds, such as glutathione, phytochelatins, or metallothioneins. We used an improved high-performance liquid chromatography method for the simultaneous measurement of thiol-containing compounds from cysteine and its derivatives (γ-glutamylcysteine, glutathione) to higher-molecular-mass compounds (phytochelatins). We found that glutathione and γ-glutamylcysteine contents increased when the ectomycorrhizal fungus Paxillus involutus was exposed to cadmium. An additional compound with a 3-kDa molecular mass, most probably related to a metallothionein, increased drastically in mycelia exposed to cadmium. The relative lack of phytochelatins and the presence of a putative metallothionein suggest that ectomycorrhizal fungi may use a different means to tolerate heavy metals, such as Cd, than do their plant hosts.     

Alteration of thiol pools in roots and shoots of maize seedlings exposed to cadmium : adaptation and developmental cost

Roots of intact 5-day-old maize (Zea mays L.) seedlings were exposed to 3 micromolar Cd during a 7-day period. Cysteine, γ-glutamylcysteine, glutathione (GSH), and Cd-induced acid-soluble thiols (ASTs), including phytochelatins, were quantified in roots and shoots. Adaptation to Cd and its cost to seedling development were evaluated by measuring Cd content, tissue fresh weight, and rate of root elongation. Roots contained 60 to 67% of the Cd in the seedlings between 4 and 7 days of exposure. Exposure to Cd decreased the fresh weight gain in roots from day 4 onward without affecting the shoots. Between days 1.5 and 3.5 of Cd treatment, roots elongated more slowly than controls; however, their growth rate recovered thereafter and exceeded that of controls. Exposure to Cd did not appreciably affect the concentration of cysteine in the seedlings. However, the initial low concentration of γ-glutamylcysteine increased (after a lag of 6 hours in roots and 2 days in shoots), reaching a plateau by day 6 at 28.5 nanomoles per gram of fresh weight in roots and by day 5 at 19.1 nanomoles per gram of fresh weight in shoots. During the first 9 hours of Cd exposure, the concentration of GSH in roots decreased dramatically (at 31.6 nanomoles per gram of fresh weight per hour) and thereafter decreased more slowly than in controls. The depletion of GSH in the roots (366 nanomoles per gram of fresh weight) matched the synthesis of ASTs (349 nanomoles per gram of fresh weight) during the first 48 hours. The concentration of ASTs in roots increased steadily thereafter to reach 662.2 nanomoles per gram of fresh weight by 6 days of Cd exposure. In shoots, Cd had little influence on the concentration of GSH, but ASTs still accumulated to 173.3 nanomoles per gram fresh weight after 5 days. The molar ratio of thiols in ASTs to Cd increased to a maximum of 10.24 in roots after 4 hours and of 4.25 in shoots after 2 days of Cd exposure. After 4 days, the ratio reached a plateau of approximately 2 in roots and between 2 and 3 in shoots, as if a steady state of Cd chelation had been achieved in both organs. The plateau coincided with recovered root elongation or an adaptation to Cd. The reduced fresh weight gain of the roots during this time, however, indicated that the synthesis of Cd-induced thiols was at a cost to root development.     

Characterization of the glutathione conjugate of the semisynthetic flavonoid monoHER

Flavonoids protect against oxidative stress by scavenging free radicals. During this protection flavonoids are oxidized. The oxidized flavonoids formed are often reactive. Consequently, protection by flavonoids can result in the formation of toxic products. In this study the oxidation of 7-mono-O-(β-hydroxyethyl)rutoside (monoHER), which is a constituent of the registered drug Venoruton, was studied in the absence and presence of glutathione (GSH). MonoHER was oxidized by horseradish peroxidase/H₂O₂. Spectrophotometric and HPLC analysis showed that in the presence of GSH, a monoHER–GSH conjugate was formed, which was identified as 2′-glutathionyl monohydroxyethylrutoside by mass spectrometric analysis and ¹H NMR. Preferential formation of this glutathione adduct in the B ring at C2′ was confirmed by molecular quantum chemical calculations. This conjugate was also detected in the bile fluid of a healthy volunteer after iv administration of monoHER, demonstrating its formation in vivo. These results indicate that in the process of offering protection against free radicals, monoHER is converted into an oxidation product that is reactive toward thiols. The formation of this thiol-reactive oxidation product is potentially harmful. Thus, the supposed beneficial effect of monoHER as an antioxidant may be accompanied by the formation of products with an electrophilic, toxic potential.     

Destabilization and denaturation of cellular protein by glutathione depletion

This investigation tested the hypothesis that depletion of intracellular glutathione, in contrast to its oxidation, could lead to non-native oxidation of protein thiols, thereby trapping proteins in an unstable conformation. Chinese hamster cells were exposed to the α,β-unsaturated dicarboxylic acid diethylmaleate in order to produce rapid gluthathione (GSH) depletion without oxidation. Measurement of the fluorescence of oxidized 2′,7′-dichlorofluorescein diacetate indicated that reactive oxygen species accumulated in GSH depleted cells. Glutathione depletion was found to alter protein thiol/disulfide exchange ratios such that 17 to 23 nmol of protein SH/mg protein underwent oxidation. Differential scanning calorimetry (DSC) of glutathione depleted cells yielded a profile of specific heat capacity versus temperature that was characteristic of cells containing destabilized and denatured protein. In addition, cells depleted of glutathione exhibited a two-fold increase in NP-40 insoluble protein. These results indicate that depletion of intracellular glutathione caused oxidation of protein thiols, protein denaturation and aggregation and provide a mechanism to explain how GSH depletion can initiate stress responses.     

Glutathione Reductase-null Malaria Parasites Have Normal Blood Stage Growth but Arrest during Development in the Mosquito

Malaria parasites contain a complete glutathione (GSH) redox system, and several enzymes of this system are considered potential targets for antimalarial drugs. Through generation of a γ-glutamylcysteine synthetase (γ-GCS)-null mutant of the rodent parasite Plasmodium berghei, we previously showed that de novo GSH synthesis is not critical for blood stage multiplication but is essential for oocyst development. In this study, phenotype analyses of mutant parasites lacking expression of glutathione reductase (GR) confirmed that GSH metabolism is critical for the mosquito oocyst stage. Similar to what was found for γ-GCS, GR is not essential for blood stage growth. GR-null parasites showed the same sensitivity to methylene blue and eosin B as wild type parasites, demonstrating that these compounds target molecules other than GR in Plasmodium. Attempts to generate parasites lacking both GR and γ-GCS by simultaneous disruption of gr and γ-gcs were unsuccessful. This demonstrates that the maintenance of total GSH levels required for blood stage survival is dependent on either de novo GSH synthesis or glutathione disulfide (GSSG) reduction by Plasmodium GR. Our studies provide new insights into the role of the GSH system in malaria parasites with implications for the development of drugs targeting GSH metabolism.     

Glutathione monoethyl ester: high-performance liquid chromatographic analysis and direct preparation of the free base form

Glutathione monoethyl ester (L-gamma-glutamyl-L-cysteinylglycine ethyl ester) was shown by R. N. Puri and A. Meister (1983, Proc. Natl. Acad. Sci. USA 80, 5258-5260) to be taken up by several tissues and intracellularly hydrolyzed to GSH. Since GSH itself is not significantly taken up by tissues, glutathione monoesters provide the most direct and convenient means available for increasing the intracellular GSH concentration of many tissues and cell types. In previous studies glutathione esters were prepared by HCl- or H2SO4-catalyzed esterification, and the product esters were precipitated as acidic salts by addition of ether to the reaction mixtures. In the present studies, glutathione monoethyl ester was synthesized by H2SO4-catalyzed esterification in the presence of sodium sulfate as the dehydrating agent. When no GSH remained, alcohol-washed Dowex-1 resin (hydroxide form) was added to remove sulfate and neutralize the reaction mixture. After the resin was removed by filtration, glutathione monoethyl ester crystallized in the chilled filtrate. The product was free of sulfate, GSH, and glutathione diester; its solutions in water or saline were neutral. Preparations obtained to date are nontoxic when administered to mice in doses up to at least 10 mmol/kg. Progress of the esterification reaction and purity of the product were determined quantitatively by HPLC after derivatization of the thiols with monobromobimane. Elution times of GSH, glutathione diester, and glutathione monoesters involving either the glutamyl or the glycyl carboxylate groups are reported.